CN102334606A - Preparation method for lactic acid-producing bacillus subtilis microbial preparation - Google Patents
Preparation method for lactic acid-producing bacillus subtilis microbial preparation Download PDFInfo
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Abstract
The invention discloses a preparation method for lactic acid-producing bacillus subtilis microbial preparation. After undergoing primary fermentation and secondary fermentation, a seed culture solution is then transferred into a bran solid medium, and is fermented under appropriate conditions, and thereby the lactic acid-producing bacillus subtilis microbial preparation is prepared. The preparation method optimizes a fermentation medium, a thallus as the seed culture solution, more than 80 percent of which are formed into spores, is inoculated into the fermentation medium, and is subjected to the primary fermentation and the secondary fermentation under the appropriate conditions, a secondary seed solution is then inoculated into the bran solid medium, and is controlled to ferment at high density under appropriate conditions, so that a large quantity of spores can be formed, and meanwhile, the affection of foreign bacteria is prevented. The preparation method effectively solves the problems of the bran fermentation process in the prior art, such as excessive foreign bacteria, uneven ventilation and heat dissipation, non-constant temperature and humidity and the like, and greatly increases the viable count of the bacilli subtilis; in a test, the viable count per gram of the lactic acid-producing bacillus subtilis microbial preparation prepared by the method is 20 to 25 billion, the spore rate is larger than 80 percent, and the foreign bacteria rate is 0.5 to 2 percent.
Description
Technical field:
The invention belongs to microorganism field, be specifically related to a kind of preparation method of lactic acid producing bacillus subtilis bacteria microorganism preparation.
Background technology:
Bacillus subtilis (Bacillus subtilis) is that China Ministry of Agriculture allows one of bacillus as feed addictive, and it is developed into the microorganism fodder preparation more and more.Bacillus subtilis has very strong protease, lipase, amylase isoreactivity, can produce antibiotic, in animal intestinal, has than Johnson & Johnson's thing and takes the oxygen ability by force, can under anaerobic produce L-lactic acid.These characteristics promote growth to play an important role to promoting zootrophic feed conversion rate and diseases prevention of digesting and assimilating, improving animal.
Therefore in feed, adding the bacillus subtilis that can produce L-lactic acid is a very selection of green health.
Preparation bacillus subtilis bacteria microorganism preparation generally adopts the solid-state traditional zymotic technology of open type wheat bran at present, adopts natural wind, natural temperature, humidity, and in the bacillus subtilis bacteria microorganism preparation of its preparation, assorted bacterium rate is high, and the viable count fluctuation is big.
Summary of the invention:
The preparation method who the purpose of this invention is to provide the lactic acid producing bacillus subtilis bacteria microorganism preparation that a kind of assorted bacterium rate is low, viable count is high.
The preparation method of lactic acid producing bacillus subtilis bacteria microorganism preparation of the present invention is characterized in that, may further comprise the steps:
(1): bacillus subtilis (Bacillus subtilis) activation, 80% above thalline formation gemma is changed in the sterilized water, form seed culture fluid, its concentration is 5.0 * 10
8Cfu/ml～2.0 * 10
9Cfu/ml;
(2): seed culture fluid is transferred in the fermentation tank that contains the one grade fermemtation culture medium with 3%～5% inoculum concentration; 37 ± 1 ℃ of fermentation temperatures; Stir speed (S.S.) 200～350rpm, throughput 0.8～0.9V/Vmin, oxyty 85～90%; Fermentation time is 8～16 hours, processes primary seed solution;
(3): primary seed solution is transferred to 2～10% inoculum concentration carries out second order fermentation in the fermentation tank that contains the second order fermentation culture medium; 37 ± 1 ℃ of fermentation temperatures, stir speed (S.S.) 150～250rpm, throughput 0.8～0.9V/Vmin; Oxyty 85～90%; Fermentation time 12～30h makes bacillus subtilis grow to exponential phase, processes secondary seed solution;
(4): in gnotobasis; Secondary seed solution with in 3～5% the inoculum concentration access wheat bran solid medium, is mixed, and the control ventilation is: 3～10 rates of ventilation/hour; 30～40 ℃ of temperature; Cultivated 60～72 hours under humidity 80%～90% condition, stir the wheat bran solid medium between culture period, prepare lactic acid producing bacillus subtilis bacteria microorganism preparation thus;
Described firsts and seconds fermentation medium all is: every liter contains MgSO
40.4～1.0g, KH
2PO
40.5～3g, K
2HPO
40.5～3g, NaCl3～10g, peptone 5～20g, beef extract 3～10g, surplus is a water, pH7.2 ± 0.3;
Described wheat bran solid medium is: by gross mass mark 100%, be made up of wheat bran 48%～60%, ammonium sulfate 0.1%～0.5%, ammonium chloride 0.1%～0.5% and water 39%～51%.
Described bacillus subtilis (Bacillus subtilis) activation, that form gemma more than 80% is changed in the sterilized water; Preferably bacillus subtilis is inoculated into beef extract-peptone agar medium test tube slant; Cultivated 12～16 hours for 37 ℃, and then be inoculated into beef extract-peptone agar medium eggplant bottle inclined-plane, 37 ℃ when being cultured to 80% above thalline formation gemma; Change over to again in the sterilized water, generally just can reach in 24～30 hours 37 ℃ of cultivations.
Stirring the wheat bran solid medium between the culture period in the described step (4) preferably whenever stirred once at a distance from 3～5 hours.
The lactic acid producing bacillus subtilis bacteria microorganism preparation that process preparation method of the present invention prepares can be packed, after finished product detection is qualified, as commodity selling after 55～60 ℃ of dried.
Beef extract-peptone agar medium of the present invention belongs to conventional culture medium of the prior art, and its prescription is: every liter contains peptone 10g, beef extract 5g, NaCl 5g, agar 15g～20g, and surplus is a water, pH7.2～7.4.
Beneficial effect of the present invention is following:
The present invention has optimized fermentation medium; The thalline that forms gemma more than 80% is inoculated in the fermentation medium as seed culture fluid; Under appropriate condition, carry out the firsts and seconds fermentation, be inoculated in the wheat bran solid medium with secondary seed solution again, control it and under appropraite condition, carry out highdensity fermentation; Form a large amount of gemma, avoid the influence of assorted bacterium simultaneously.The solution that the present invention is favourable in the prior art the assorted bacterium in the wheat bran sweat too much, ventilate, problem such as heat radiation inequality, temperature, humidity are non-constant; Improved the viable count of bacillus subtilis greatly; Through detecting, according to the lactic acid producing bacillus subtilis bacteria microorganism preparation that the inventive method prepares, its every gram microorganism formulation contains viable count 200～25,000,000,000 viable bacteria; Gemma rate＞80%, assorted bacterium rate 0.5%～2%.
Bacillus subtilis of the present invention (Bacillus subtilis) is the bacterial strain that often uses in the prior art, and it discloses in a lot of patent documentations and non-patent literature, like non-patent literature (Xu Guohuan; Wu Yue the lady in the moon, Fu Tianxi etc., microorganism formulation is to the influence of Sarotherodon sp growth and feed apparent digestibility; China's feed; 2008.21:26-28), this bacterial strain the applicant also hold, and guarantee in 20 years applyings date, to provide to the public.
Description of drawings:
Fig. 1 is the bacillus subtilis growth curve chart in the one grade fermemtation among the embodiment 1;
Fig. 2 is the bacillus subtilis growth curve chart in the second order fermentation among the embodiment 1.
The specific embodiment:
Below be to further specify to of the present invention, rather than limitation of the present invention.
Embodiment 1:
Picking one ring thalline is seeded to the beef extract-peptone agar test tube slant culture medium on the freeze-drying kind of bacillus subtilis, cultivates 16 hours for 37 ℃; The test tube slant seed is transferred in the beef extract-peptone agar bottle inclined plane culture medium of eggplant again, cultivated 24 hours for 37 ℃, through detecting, it forms gemma more than 80%.With sterilized water ripe bacterium mud on the eggplant bottle is scraped to wash and, the 250ml triangular flask of packing into, vibration makes thalline even, forms seed culture fluid, and its concentration is 1.2 * 10
9Cfu/ml.The prescription of described beef extract-peptone agar medium is: every liter contains peptone 10g, beef extract 5g, NaCl 5g, agar 15g～20g, and surplus is a water, pH7.2～7.4,121 ℃ autoclaving 20min.
Seed culture fluid changed in the 30L seeding tank that the one grade fermemtation culture medium is housed with 5% inoculum concentration carry out fermented and cultured, liquid amount 70%, 37 ± 1 ℃ of fermentation temperatures; Stir speed (S.S.) 200rpm, throughput 0.8～0.9V/Vmin, oxyty 85～90%; The light absorption value of 600nm is once surveyed in every 2h sampling from the inoculation back; As blank, make bacillus subtilis growth curve chart (Fig. 1) with nonvaccinated culture medium, can know that by curve bacillus subtilis sharply increases at 8～12h bacterium number; Show that thalline has arrived exponential phase, the bacterium liquid of taking fermentation time and be 8 hours is processed primary seed solution; With 10% inoculum concentration, further enlarged culture, liquid amount 70% are gone into to be equipped with in the 300L fermentation tank of second order fermentation culture medium in switching with primary seed solution; 37 ± 1 ℃ of fermentation temperatures, stir speed (S.S.) 250rpm, throughput 0.8～0.9V/Vmin; Oxyty 85～90%; The light absorption value of 600nm is once surveyed in every 2h sampling from the inoculation back, as blank, makes bacillus subtilis growth curve chart (Fig. 2) with nonvaccinated culture medium; Can know that by curve bacillus subtilis sharply increases at 12～25h bacterium number; Therefore show that thalline has reached exponential phase, take the bacterium liquid in this period to obtain secondary seed solution that what choose in the present embodiment is that fermentation time is that 16 hours bacterium liquid is as secondary seed solution.Described firsts and seconds fermentation medium is, and every liter contains MgSO
40.5g, KH
2PO
41g, K
2HPO
41g, NaCl 5g, peptone 10g, beef extract 5g, surplus is a water, pH7.0 is soluble in water with above-mentioned substance, again 121 ℃ of autoclaving 20min.
Before the inoculation, open ozone generator and the uviol lamp switch of cultivating the room earlier, cultivate room area: 25m
2, sterilization 30min, preparation gnotobasis; In this gnotobasis, secondary seed solution is inoculated in the wheat bran solid medium with 3% inoculum concentration, mixes, the ventilation in the control wheat bran solid medium is: 10 rates of ventilation/hour; 30～40 ℃ of temperature; Humidity is to cultivate 60 hours under 80～90% the condition, whenever stirs wheat bran solid culture base-material once at a distance from 3 hours between culture period, makes lactic acid producing bacillus subtilis bacteria microorganism preparation therefrom.Described wheat bran solid medium, every kilogram contains wheat bran 500g, ammonium sulfate 4g, ammonium chloride 2g, water 494g.Its collocation method is that ammonium chloride and ammonium sulfate are mixed with all the other solid materials with water-soluble earlier again, stirs.Rotating digester or meal braizing machine then, pressure, temperature is respectively: 1.05～1.3Kg/cm
3, under 121～125 ℃ of conditions, sterilization 30～40min is cooled to 40～50 ℃ of shallow basins of packing, and 5 kilograms in every basin is subsequent use.
One, the detection of viable count and assorted bacterium rate:
1, the method for plate culture count
1.1, the lactic acid producing bacillus subtilis bacteria microorganism preparation 10g that takes by weighing present embodiment is as sample, puts into the conical flask that fills the 90ml sterilized water and have bead, the shaking table 200rpm 20min that vibrates leaves standstill 20～30S, processes sample suspension.
1.2, draw the 1mL sample suspension with the 1ml aseptic straw, put into the test tube that fills the 9mL aqua sterilisa, shake well makes and mixes, and promptly processes 1: 100 even dilution.
1.3, draw 1: 100 dilution 1mL with the 1mL aseptic straw, inject slowly along tube wall and contain the 9mL aqua sterilisa in vitro, the jolting test tube mixes, and makes 1: 1000 dilution.Other gets 1mL sterilization suction pipe, presses the aforesaid operations order, does 10 times and increases progressively dilution, processes 10 respectively
-4, 10
-5, 10
-6... 10
-9Gradient dilution liquid so whenever increases progressively dilution once, promptly uses 1 1mL sterilization suction pipe instead.
1.4, select 2-3 suitable dilution factor (generally to get 10
-6, 10
-7, 10
-8), with sterilization 100 μ L micropipettors, respectively to get 0.1mL and be inoculated in the beef extract-peptone agar culture plate that has prepared, each dilution factor is made 3 flat boards, evenly is applied to whole surface with aseptic push rod.
1.5, with plate be inverted in cultivate 48h ± 2h in 37 ℃ ± 1 ℃ constant incubator after, choose the plate count of clump count between 30-300.
2, bacillus subtilis colonial morphology
Morphological feature: thalline is shaft-like, and the bacterium colony rough surface is opaque, and fold is arranged, dirty white or little yellow, bacterium colony irregular cycle.
3, colony counting
3.1, dilution selection
3.1.1, should select the dilution factor of average bacterium colony between 30-300, multiply by the extension rate report.
3.1.2, if two dilution factors are arranged.The bacterium colony number average of its growth is then looked and how to be decided between the two if it than being less than or equal to 2, should report its average between 30-300; If greater than 2, then report wherein less numeral.
3.1.3, if all dilution average bacterium colony number averages greater than 300.Then should multiply by the extension rate report by dilution factor the highest average clump count.
3.1.4, if the average bacterium colony number average of all dilution factors less than 30, then should multiply by the extension rate report by dilution factor minimum average clump count.
3.2, colony counting and discriminating
3.2.1, make a distinction according to the colony characteristics and the assorted bacterium of the bacterial classification of examined product.Carry out Gram or spore staining in case of necessity, microscopically is observed.Effectively other bacterium colonies beyond the bacillus subtilis bacterium colony are assorted bacterium clump count.
3.2.2, should select the dilution flat board of average bacterium colony between 30-300,5 target bacterium colonies of picking carry out Physiology and biochemistry by " uncle Jie Shi bacterial system identification handbook " the 8th edition, " common bacteria system identification handbook " and identify at random.If the result meets table 1 characteristic:
Table 1
Can judge that then this bacterium colony is effective bacillus subtilis bacterium colony.
4, calculate:
Bacillus subtilis viable count (CFU/mL)=three flat-plate bacterial colony average * extension rate * 10 * n/5-n of same dilution factor are effective bacterial count of bacillus subtilis
Assorted bacterium rate (%)=assorted bacterium sum/(bacillus subtilis viable count+assorted bacterium sum)
With the discovery of testing of the lactic acid producing bacillus subtilis bacteria microorganism preparation of present embodiment, its every gram contains 25,000,000,000 viable bacterias of viable count (gemma rate＞80%), assorted bacterium rate 1%.
Bacillus subtilis anaerobic fermentation lactic acid producing: the bacillus subtilis spore in the lactic acid producing Bacillus subtillis microorganism formulation is inserted the wheat bran solid medium, and inoculum concentration is weight percentage: 2-4%.Temperature 30-40 ℃, anaerobism was cultivated 40-70 hour.The fermentation solid content that will pass through above-mentioned anaerobic fermentation is soluble in water, is heated to 60～100 ℃, the centrifugal 10～15min of 5000rpm in centrifuge.Get supernatant and measure the L-lactic acid content, record the L-lactic acid content and reach more than the 1.0g/L with L-lactic dehydrogenase enzyme process.
This lactic acid producing bacillus subtilis bacteria microorganism preparation through 55～60 ℃ of vacuum drying treatment, is packaged into finished product, detect qualified after, as commodity selling.
Embodiment 2:
Picking one ring thalline is seeded to the beef extract-peptone agar test tube slant culture medium on the freeze-drying kind of bacillus subtilis, cultivates 12 hours for 37 ℃; The test tube slant seed is transferred in the beef extract-peptone agar bottle inclined plane culture medium of eggplant again, cultivated 30 hours for 37 ℃, through detecting, its thalline more than 80% forms gemma.With sterilized water ripe bacterium mud on the eggplant bottle is scraped to wash and, the 250ml triangular flask of packing into, vibration makes thalline even, forms seed culture fluid, and its concentration is 2.0 * 10
9Cfu/ml.The prescription of described beef extract-peptone agar medium is: every liter contains peptone 10g, beef extract 5g, NaCl 5g, agar 15g～20g, and surplus is a water, pH7.2～7.4,121 ℃ autoclaving 20min.
Seed culture fluid changed in the 30L seeding tank that the one grade fermemtation culture medium is housed with 3% inoculum concentration carry out fermented and cultured; Liquid amount 70%, 37 ± 1 ℃ of fermentation temperatures, stir speed (S.S.) 350rpm; Throughput 0.8～0.9V/Vmin; Oxyty 85～90%, fermentation time is 16 hours, processes primary seed solution; With 10% inoculum concentration, further enlarged culture, liquid amount 70% are gone into to be equipped with in the 300L fermentation tank of second order fermentation culture medium in switching with primary seed solution; 37 ± 1 ℃ of fermentation temperatures, stir speed (S.S.) 150rpm, throughput 0.8～0.9V/Vmin; Oxyty 85～90%; Fermentation time is 30 hours, has reached exponential phase through detecting thalline, obtains secondary seed solution thus.Described firsts and seconds fermentation medium is that every liter contains MgSO
40.4g, KH
2PO
43g, K
2HPO
40.5g, NaCl 10g, peptone 5g, beef extract 10g, surplus is a water, pH7.4 is soluble in water with above-mentioned substance, again 121 ℃ of autoclaving 20min.
Before the inoculation, open ozone generator and the uviol lamp switch of cultivating the room earlier, cultivate room area: 25m
2, sterilization 30min, preparation gnotobasis; In this gnotobasis, secondary seed solution is inoculated in the wheat bran solid medium with 4% inoculum concentration, mixes, the ventilation in the control wheat bran solid medium is: 3 rates of ventilation/hour; 30～40 ℃ of temperature; Humidity is to cultivate 72 hours under 80～90% the condition, whenever stirs wheat bran solid culture base-material once at a distance from 5 hours between culture period, makes lactic acid producing bacillus subtilis bacteria microorganism preparation therefrom.Described wheat bran solid medium, every kilogram contains wheat bran 480g, ammonium sulfate 5g, ammonium chloride 5g, water 510g.Its collocation method is that ammonium chloride and ammonium sulfate are mixed with all the other solid materials with water-soluble earlier again, stirs.Rotating digester or meal braizing machine then, pressure, temperature is respectively: 1.05～1.3Kg/cm
3, under 121～125 ℃ of conditions, sterilization 30～40min is cooled to 40～50 ℃ of shallow basins of packing, and 5 kilograms in every basin is subsequent use.
According to the method for embodiment 1, with the discovery of testing of the lactic acid producing bacillus subtilis bacteria microorganism preparation of present embodiment, its every gram contains 20,000,000,000 viable bacterias of viable count (gemma rate＞80%), assorted bacterium rate 0.5%.
Bacillus subtilis anaerobic fermentation lactic acid producing: the bacillus subtilis spore in the lactic acid producing Bacillus subtillis microorganism formulation is inserted the wheat bran solid medium, and inoculum concentration is weight percentage: 2-4%.Temperature 30-40 ℃, anaerobism was cultivated 40-70 hour.The fermentation solid content that will pass through above-mentioned anaerobic fermentation is soluble in water, is heated to 60～100 ℃, the centrifugal 10～15min of 5000rpm in centrifuge.Get supernatant and measure the L-lactic acid content, record the L-lactic acid content and reach more than the 1.0g/L with L-lactic dehydrogenase enzyme process.
This lactic acid producing bacillus subtilis bacteria microorganism preparation through 55～60 ℃ of vacuum drying treatment, is packaged into finished product, detect qualified after, as commodity selling.
Embodiment 3:
Picking one ring thalline is seeded to the beef extract-peptone agar test tube slant culture medium on the freeze-drying kind of bacillus subtilis, cultivates 16 hours for 37 ℃; The test tube slant seed is transferred in the beef extract-peptone agar bottle inclined plane culture medium of eggplant again, cultivated 24 hours for 37 ℃, through detecting, it forms gemma more than 80%.With sterilized water ripe bacterium mud on the eggplant bottle is scraped to wash and, the 250ml triangular flask of packing into, vibration makes thalline even, forms seed culture fluid, and cell concentration is 5.0 * 10 in the seed liquor
8Cfu/ml.The prescription of described beef extract-peptone agar medium is: every liter contains peptone 10g, beef extract 5g, NaCl 5g, agar 15g～20g, and surplus is a water, pH7.2～7.4,121 ℃ autoclaving 20min.
Seed culture fluid changed in the 30L seeding tank that the one grade fermemtation culture medium is housed with 4% inoculum concentration carry out fermented and cultured, liquid amount 70%, 37 ± 1 ℃ of fermentation temperatures; Stir speed (S.S.) 250rpm; Throughput 0.8～0.9V/Vmin, oxyty 85～90%, fermentation time is 12 hours; Show that through detecting thalline has arrived exponential phase, processes primary seed solution; With 2% inoculum concentration, further enlarged culture, liquid amount 70% are gone into to be equipped with in the 300L fermentation tank of second order fermentation culture medium in switching with primary seed solution; 37 ± 1 ℃ of fermentation temperatures, stir speed (S.S.) 200rpm, throughput 0.8～0.9V/Vmin; Oxyty 85～90%; Fermentation time is 12 hours, shows that through detecting thalline has reached exponential phase, obtains secondary seed solution thus.Described firsts and seconds fermentation medium is that every liter contains MgSO
41g, KH
2PO
40.5g, K
2HPO
43g, NaCl 3g, peptone 20g, beef extract 3g, surplus is a water, pH7.0 is soluble in water with above-mentioned substance, again 121 ℃ of autoclaving 20min.
Before the inoculation, open ozone generator and the uviol lamp switch of cultivating the room earlier, cultivate room area: 25m
2, sterilization 30min, preparation gnotobasis; In this gnotobasis, second order fermentation liquid is inoculated in the wheat bran solid medium with 5% inoculum concentration, mixes, the ventilation in the control wheat bran solid medium is: 6 rates of ventilation/hour; 30～40 ℃ of temperature; Humidity is to cultivate 66 hours under 80～90% the condition, whenever stirs wheat bran solid culture base-material once at a distance from 4 hours between culture period, makes lactic acid producing bacillus subtilis bacteria microorganism preparation therefrom.Described wheat bran solid medium, every kilogram contains wheat bran 600g, ammonium sulfate 1g, ammonium chloride 5g, water 394g.Its collocation method is that ammonium chloride and ammonium sulfate are mixed with all the other solid materials with water-soluble earlier again, stirs.Rotating digester or meal braizing machine then, pressure, temperature is respectively: 1.05～1.3Kg/cm
3, under 121～125 ℃ of conditions, sterilization 30～40min is cooled to 40～50 ℃ of shallow basins of packing, and 5 kilograms in every basin is subsequent use.
According to the method for embodiment 1, with the discovery of testing of the lactic acid producing bacillus subtilis bacteria microorganism preparation of present embodiment, its every gram contains 22,000,000,000 viable bacterias of viable count (gemma rate＞80%), assorted bacterium rate 2%.
Bacillus subtilis anaerobic fermentation lactic acid producing: the bacillus subtilis spore in the lactic acid producing Bacillus subtillis microorganism formulation is inserted the wheat bran solid medium, and inoculum concentration is weight percentage: 2-4%, temperature 30-40 ℃, anaerobism was cultivated 30-70 hour.The fermentation solid content that will pass through above-mentioned anaerobic fermentation is soluble in water, is heated to 60～100 ℃, the centrifugal 10～15min of 5000rpm in centrifuge.Get supernatant and measure the L-lactic acid content, record the L-lactic acid content and reach more than the 1.0g/L with L-lactic dehydrogenase enzyme process.
This lactic acid producing bacillus subtilis bacteria microorganism preparation through 55～60 ℃ of vacuum drying treatment, is packaged into finished product, detect qualified after, as commodity selling.
Claims (3)
1. the preparation method of a lactic acid producing bacillus subtilis bacteria microorganism preparation is characterized in that, may further comprise the steps:
(1): bacillus subtilis (Bacillus subtilis) activation, 80% above thalline formation gemma is changed in the sterilized water, form seed culture fluid, its concentration is 5.0 * 10
8Cfu/ml～2.0 * 10
9Cfu/ml;
(2): seed culture fluid is transferred in the fermentation tank that contains the one grade fermemtation culture medium with 3%～5% inoculum concentration; 37 ± 1 ℃ of fermentation temperatures; Stir speed (S.S.) 200～350rpm, throughput 0.8～0.9V/Vmin, oxyty 85～90%; Fermentation time is 8～16 hours, processes primary seed solution;
(3): primary seed solution is transferred to 2～10% inoculum concentration carries out second order fermentation in the fermentation tank that contains the second order fermentation culture medium; 37 ± 1 ℃ of fermentation temperatures, stir speed (S.S.) 150～250rpm, throughput 0.8～0.9V/Vmin; Oxyty 85～90%; Fermentation time 12～30h makes bacillus subtilis grow to exponential phase, processes secondary seed solution;
(4): in gnotobasis; Secondary seed solution with in 3～5% the inoculum concentration access wheat bran solid medium, is mixed, and the control ventilation is: 3～10 rates of ventilation/hour; 30～40 ℃ of temperature; Cultivated 60～72 hours under humidity 80%～90% condition, stir the wheat bran solid medium between culture period, prepare lactic acid producing bacillus subtilis bacteria microorganism preparation thus;
Described firsts and seconds fermentation medium all is: every liter contains MgSO
40.4～1.0g, KH
2PO
40.5～3g, K
2HPO
40.5～3g, NaCl 3～10g, peptone 5～20g, beef extract 3～10g, surplus is a water, pH7.2 ± 0.3;
Described wheat bran solid medium is: by gross mass mark 100%, be made up of wheat bran 48%～60%, ammonium sulfate 0.1%～0.5%, ammonium chloride 0.1%～0.5% and water 39%～51%.
2. the preparation method of lactic acid producing bacillus subtilis bacteria microorganism preparation according to claim 1; It is characterized in that; Described bacillus subtilis (Bacillus subtilis) activation, that form gemma more than 80% is changed in the sterilized water is that bacillus subtilis is inoculated into beef extract-peptone agar medium test tube slant; Cultivated 12～16 hours for 37 ℃; And then be inoculated into beef extract-peptone agar medium eggplant bottle inclined-plane, 37 ℃ when being cultured to 80% above thalline and forming gemma, change in the sterilized water again.
3. the preparation method of lactic acid producing bacillus subtilis bacteria microorganism preparation according to claim 1 is characterized in that, stirring the wheat bran solid medium between the culture period in the described step (4) is whenever to stir once at a distance from 3～5 hours.
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| CN105039213A (en) * | 2015-07-09 | 2015-11-11 | 广东碧德生物科技有限公司 | Aerobic denitrifying bacillus subtilis preparation and preparation method thereof |
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| CN108179130A (en) * | 2018-03-21 | 2018-06-19 | 广州同心源生物科技有限公司 | A kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder |
| CN111499444A (en) * | 2020-03-13 | 2020-08-07 | 广西壮族自治区农业科学院 | Preparation method and application of activated amino acid |
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| CN104450585A (en) * | 2014-12-16 | 2015-03-25 | 四川省农业科学院农产品加工研究所 | Bacillus subtilis for preparing L-lactic acid with high yield by taking corn steep liquor as only nitrogen source |
| CN104450585B (en) * | 2014-12-16 | 2017-05-03 | 四川省农业科学院农产品加工研究所 | Bacillus subtilis for preparing L-lactic acid with high yield by taking corn steep liquor as only nitrogen source |
| CN105039213A (en) * | 2015-07-09 | 2015-11-11 | 广东碧德生物科技有限公司 | Aerobic denitrifying bacillus subtilis preparation and preparation method thereof |
| CN105941835A (en) * | 2016-06-30 | 2016-09-21 | 河南省岳氏精忠科技有限公司 | Method for preparing bacillus subtilis traditional Chinese medicine feed additive |
| CN106107033A (en) * | 2016-06-30 | 2016-11-16 | 河南省岳氏精忠科技有限公司 | A kind of preparation method of feeding bacillus subtilis Chinese medicine mycopowder |
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| CN111499444A (en) * | 2020-03-13 | 2020-08-07 | 广西壮族自治区农业科学院 | Preparation method and application of activated amino acid |
| CN114437984A (en) * | 2022-02-18 | 2022-05-06 | 鑫九通科技(武汉)有限公司 | Preparation method of lactic acid-producing bacillus coagulans microbial preparation |
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